Hirudin: The Promising Antithrombotic

Abstract

It has long been known that medicinal leeches (Hirudo medicinalis) contain a substance with anticoagulant properties. In early studies, aqueous extracts and dry preparations from leeches containing minute amounts of the active substance were used to prevent blood from clotting. Only after the development of protein chemistry and the elucidation of the biochemistry of blood coagulation was the anticoagulant agent, hirudin, isolated and its chemical nature and mode of action clarified.The historical background of hirudin isolation as well as the individual steps of its biochemical and pharmacological characterization have been described in several reviews (36,68,70,72,74,79). BIOCHEMISTRYHirudin is extracted from the homogenized heads of medicinal leeches and enriched by precipitation procedures followed by ion-exchange chromatography and gel filtration (2,65,98,137). Affinity chromatography on matrix-bound thrombin can be used to obtain highly purified hirudin preparations (139,140). The pure anticoagulant agent obtained is a carboxyhydrate-free single chain miniprotein containing 65 amino acids with a molecular weight of about 7 kDa. The amino acid composition is characterized by a remarkably high content of acidic amino acids at the C-terminal, the absence of arginine, methionine, and tryptophan, and the presence of a sulfated tyrosine residue and three intramolecular disulfide bridges (62,65,70).Considerable progress in the isolation, purification, and analysis of hirudin has shown the medicinal leech generates several variants of the miniprotein in trace amounts. The isoinhibitor forms share 85-90% amino acid sequence homology. Their main structural elements, which include N-terminal hydrophobic amino acids, followed by a core region linked by three disulfide bridges and the unique clustering of acidic amino acid residues in the C-terminal portion remain constant (4,17,18,113). The N-terminal amino acids Val-Val can be replaced by Ile-Thr (Fig. 1).

Comparison of in vitro and in vivo Properties of rHirudin (HBW 023) and a Synthetic Analogous Peptide

“…With regard to aPTT prolonga tion, rhirudin shows two-phase kinetics, with a ti/j a, p of, on average, 0.5 and 67.9 min respectively. These findings are in good agree ment with recently published results [2,24], The hirudin analogue, however, shows onephase kinetics, with a ty2 of 5 min.…”

“…Surprising ly, an analogous bivalent inhibitor, which consists of a peptide corresponding to the carboxy terminal region (amino acids 53-64) of hirudin linked to a Z)-Phe-Pro-Arg-Pro pep tide via a (Gly)4-residue at the amino-termi nal end, demonstrated significant anticoagu latory ability, despite the fact that the individ ual components mediate only relatively weak inhibitory effects. Other authors have already reported in detail on the biochemical and bio logical properties of both substances [2,7,11], In a series of in vitro and in vivo test systems the superiority of both inhibitors over heparins was demonstrated [ 13,[27][28][29]. However, there are only few comparative studies of rhi rudin and this analogue [12,13].…”

“…Moreover, the effi cacy of heparin and heparin derivatives is dependent on the availability of cofactors, especially antithrombin III, whose access to thrombus-bound thrombin is limited [1], During the search for more effective and tol erable antithrombotic substances hirudin was discovered, a polypeptide from the salivary glands of the leech Hirudo medicinalis. De spite the fact that the anticoagulant properties of hirudin have been known for some time, the production of the amounts required for therapeutic purposes was not possible until gene technological methods could be em ployed [2], Promising results achieved in in vitro tests could be confirmed in numerous animal models of thrombosis prophylaxis and therapy [3], Extensive interactions with thrombin affect the specificity of hirudin and its affinity for this coagulation protease and makes it independent of cofactors. Further more, hirudin is capable of inhibiting fibrinbound thrombin and thus stops thrombus growth.…”

Endothelial cells stimulated with tumor necrosis factor-alpha express varying amounts of tissue factor resulting in inhomogenous fibrin deposition in a native blood flow system. Effects of thrombin inhibitors.

“…A special mixing device was used which allowed supplementation of the native blood with an antithrombotic compound under continuous flow conditions. This report describes the effects ofrecombinant (rec.) hirudin (34), heparin, and the selective, reversible thrombin in- hibitor Ro Fig. 1 is FCS, 50 ,g/ml endothelial cell growth factor, and 100 gg/ml heparin.…”

Heparin‐induced thrombocytopenia presenting with thrombosis of multiple saphenous vein grafts and myocardial infarction

“…Lepirudin inactivates thrombin by forming a tight 1:1 complex involving noncovalent interactions without the need of a cofactor. Its half-life is 1.5 hr and is excreted through the kidneys [14]. Both argatroban and lepirudin can be monitored by an activated partial thromboplastin time (aPTT).…”